The molecular interactions between axons and glial cells constitute an area of immense interest. Many diseases produce their debilitating effects by altering the structural and functional relations between axons and glial cells, which in turn affect action potential propagation and even neuronal survival. In vertebrate myelinated axons, a structural specialization, the axo-glial septate junction (SJ), forms at the paranode between myelin loops and the axonal surface. Axo-glial SJs are characterized by ladder-like electron dense structures. Our recent work shows that axo-glial SJs are also present in Drosophila nerves. In addition to structural similarities, compelling molecular homologies are observed between axo-. glial SJs in fly and those in mouse. The Drosophila proteins: Neurexin (NRX), Contactin (CONT) and Neuroglian (NRG) form a tripartite complex that localizes to axo-glial SJs. Their murine homologs NCP1, Contactin and Neurofascin 155kDa isoform (NF155) also form a complex at axo- glial SJs. Our phenotypic analyses of Drosophila neurexin and mouse NCP1 mutants show that both mutants lack SJs. In this application, we propose to use genetic, cell biological, molecular and biochemical methods in Drosophila and mouse to address the following questions: (1) When do axo-glial SJs form during embryonic development in fly; Does axo-glial SJ formation coincide with the expression of NRX, CONT and NRG? Are axo-glial SJs absent in nrx, cont and nrg mutants? (2) What is the relationship of NRX, CONT and NRG to the formation of SJs in fly? Are NRX, CONT and NRG sufficient for the formation of SJs? (3) What is the loss of function phenotype of the mouse homolog of NRG (NF155)? What role does NF155 play in the formation of paranodal axo-glial SJs? Our studies will provide new insights into the mechanisms responsible for axon-glial interactions. In the future, these studies will advance our understanding of the functional deficits that accompany demyelinating disorders.